The invention relates to 6-mercapto-cyclodextrin derivatives, to their use for the preparation of a medicament for the reversal of drug-induced neuromuscular block, and to a kit for providing neuromuscular block and its reversal.
A neuromuscular blocking agent (NMBA, also called a muscle relaxant) is routinely used during the administration of anaesthesia to facilitate endotracheal intubation and to allow surgical access to body cavities, in particular the abdomen and thorax, without hindrance from voluntary or reflex muscle movement. NMBAs are also used in the care of critically-ill patients undergoing intensive therapy, to facilitate compliance with mechanical ventilation when sedation and analgesia alone have proved inadequate, and to prevent the violent muscle movements that are associated with electroconvulsive therapy treatment.
Based on their mechanisms of action, NMBAs are divided into two categories: depolarizing and non-depolarizing. Depolarizing neuromuscular blocking agents bind to nicotinic acetylcholine receptors (nAChRs) at the neuromuscular junction in a way similar to that of the endogenous neurotransmitter acetylcholine. They stimulate an initial opening of the ion channel, producing contractions known as fasciculations. However, since these drugs are broken down only relatively slowly by cholinesterase enzymes, compared to the very rapid hydrolysis of acetylcholine by acetylcholinesterases, they bind for a much longer period than acetylcholine, causing persistent depolarization of the end-plate and hence a neuromuscular block. Succinylcholine (suxamethonium) is the best known example of a depolarizing NMBA.
Non-depolarizing neuromuscular blocking agents compete with acetylcholine for binding to muscle nAChRs, but unlike depolarizing NMBAs, they do not activate the channel. They block the activation of the channel by acetylcholine and hence prevent cell membrane depolarization, and as a result, the muscle will become flaccid. Most of the clinically-used NMBAs belong to the non-depolarizing category. These include tubocurarine, atracurium, (cis)atracurium, mivacurium, pancuronium, vecuronium, rocuronium and rapacuronium (Org 9487).
At the end of surgery or a period of intensive care, a reversal agent of NMBAs is often given to the patient to assist the recovery of muscle function. Most commonly used reversal agents are inhibitors of acetylcholinesterase (AChE), such as neostigmine, edrophonium and pyridostigmine. Because the mechanism of action of these drugs is to increase the level of acetylcholine at the neuromuscular junction by inhibiting the breakdown of acetylcholine, they are not suitable for reversal of depolarizing NMBAs such as succinylcholine. The use of AChE inhibitors as reversal agents leads to problems with selectivity, since neurotransmission to all synapses (both somatic and autonomic) involving the neurotransmitter acetylcholine is potentiated by these agents. This non-selectivity may lead to many side-effects due to the non-selective activation of muscarinic and nicotinic acetylcholine receptors, including bradycardia, hypotension, increased salivation, nausea, vomiting, abdominal cramps, diarrhoea and bronchoconstriction. Therefore in practice, these agents can be used only after or together with the administration of atropine (or glycopyrrolate) to antagonize the muscarinic effects of acetylcholine at the muscarinic receptors in the autonomic parasympathetic neuro-effector junctions (e.g. the heart). The use of a muscarinic acetylcholine receptor (mAChR) antagonist such as atropine causes a number of side-effects, e.g., tachycardia, dry mouth, blurred vision, difficulties in emptying the bladder and furthermore may affect cardiac conduction.
A further problem with anticholinesterase agents is that residual neuro-muscular activity must be present ( greater than 10% twitch activity) to allow the rapid recovery of neuromuscular function. Occasionally, either due to hyper-sensitivity of the patient or accidental overdose, administration of NMBAs can cause complete and prolonged block of neuromuscular function (xe2x80x9cprofound blockxe2x80x9d). At present, there is no reliable treatment to reverse such a xe2x80x98profound blockxe2x80x99. Attempts to overcome a xe2x80x98profound blockxe2x80x99 with high doses of AChE inhibitors has the risk of inducing a xe2x80x9ccholinergic crisisxe2x80x9d, resulting in a broad range of symptoms related to enhanced stimulation of nicotinic and muscarinic receptors.
In European Patent Application 99,306,411 (AKZO NOBEL N.V.) the use of chemical chelators (or sequestrants) as reversal agents has been disclosed. Chemical chelators capable of forming a guest-host complex for the manufacture of a medicament for the reversal of drug-induced neuromuscular block were described. The use of chemical chelators as reversal agents for NMBAs has the advantage that they are effective in reversing the action of both depolarizing and non-depolarizing NMBAs. Their use does not increase the level of acetylcholine and therefore they produce fewer side effects and none associated with the stimulation of muscarinic and nicotinic receptors seen with the AChE reversal agents. In addition, there is no need for the combined use of an AChE inhibitor and a mAChR antagonist (e.g., atropine), while the chemical chelators may further be safely employed for the reversal of xe2x80x98profound blockxe2x80x99. Examples of such chemical chelators, as disclosed in EP 99,306,411, were selected from various classes of, mostly cyclic, organic compounds which are known for their ability to form inclusion complexes with various organic compounds in aqueous solution, e.g. cyclic oligosaccharides, cyclophanes, cyclic peptides, calixarenes, crown ethers and aza crown ethers.
The cyclodextrins, 
a class of cyclic molecules containing six or more xcex1-D-glucopyranose units linked at the 1,4 positions by xcex1-linkages as in amylose, and derivatives thereof, were identified in EP 99306411 as particularly useful in the reversal of many of the commonly used neuromuscular blocking agents, or muscle relaxants, such as rocuronium, pancuronium, vecuronium, rapacuronium, mivacurium, atracurium, (cis)atracurium, succinylcholine and tubocurarine.
It has now been found that 6-mercapto-cyclodextrin derivatives having the general formula I 
wherein m is 0-7 and n is 1-8 and m+n=7 or 8;
R is (C1-6)alkylene, optionally substituted with 1-3 OH groups, or (CH2)o-phenylene-(CH2)pxe2x80x94;
o and p are independently 0-4;
X is COOH, CONHR1, NHCOR2, SO2OH, PO(OH)2, O(CH2xe2x80x94CH2xe2x80x94O)qxe2x80x94H, OH or tetrazol-5-yl;
R1 is H or (C1-3)alkyl;
R2 is carboxyphenyl;
q is 1-3;
or pharmaceutically acceptable salts thereof;
are highly active in vivo in the reversal of the action of neuromuscular blocking agents.
No protection per se is sought for the following 6-mercapto-cyclodextrin derivatives:
6-per-deoxy-6-per-(2-hydroxyethylthio)-xcex2-cyclodextrin and
6-per-deoxy-6per-(2-hydroxyethylthio)-xcex3-cyclodextrin, which are described by Ling, C. and Darcy, R. (J. Chem. Soc. Chem Comm. 1993, (2), 203-205);
6-mono-deoxy-6-mono-(2-hydroxyethylthio)-xcex2-cyclodextrin, which is disclosed by Fujita, K. et al. (Tetr. Letters 21, 1541-1544, 1980);
6-per-deoxy-6-per-(carboxymethylthio)-xcex2-cyclodextrin, which is described by Guillo, F. et al. (Bull. Chem. Soc. Chim. Fr. 132 (8), 857-866, 1995);
6-mono-deoxy-6-mono-(carboxymethylthio)-xcex2-cyclodextrin, which is described by Akiie, T. et al. (Chem. Lett. 1994 (6), 1089-1092);
6A,6B-dideoxy-6A,6B-bis[(o-carboxyphenyl)thio]-xcex2-cyclodextrin and 6A,6B-dideoxy-6A,6B-bis(carboxymethylthiol)-xcex2-cyclodextrin, which are described by Tubashi, I. et al. (J. Am. Chem. Soc. 108, 4514-4518, 1986; and
6-per-deoxy-6-per-(2,3dihydroxypropylthio)-xcex2-cyclodextrin, which is described by Baer, H. H. and Santoyo-Gonzxc3xa1lez, F. (Carb. Res. 280, 315-321, 1996). These prior art 6-mercapto-cyclodextrin derivatives have been described in relation with different utilities in each instance.
However, the above mentioned prior art 6-mercapto-cyclodextrin derivatives do belong to the main aspect of the present invention which relates to the use of a 6-mercapto-cyclodextrin derivative according to the general formula I for the manufacture of a medicament for the reversal of drug-induced neuromuscular block.
In one embodiment the invention relates to 6-mercapto-cyclodextrin derivatives having the general formula I,
wherein m is 0-7 and n is 1-8 and m+n=7 or 8;
X is COOH, OH or CONHCH3;
R is (C1-6)alkylene or (CH2)o-phenylene-(CH2)p;
o and p are independently 0-4; or a pharmaceutically acceptable salt thereof, with the exclusion of
6-per-deoxy-6-per-(2-hydroxyethylthio)-xcex2-cyclodextrin;
6-mono-deoxy-6-mono-(2-hydroxyethylthio)-xcex2-cyclodextrin;
6-per-deoxy-6-per-(2-hydroxyethylthio)-xcex3-cyclodextrin;
6-per-deoxy-6-per-(carboxymethylthio)-xcex2-cyclodextrin;
6-mono-deoxy-6-mono-(carboxymethylthio)-xcex2-cyclodextrin;
6A,6B-dideoxy-6A,6B-bis[(o-carboxyphenyl)thio]-xcex2-cyclodextrin; and
6A,6B-dideoxy-6A,6B-bis(carboxymethylthiol)-xcex2-cyclodextrin.
The term (C1-6)alkylene as used in the definition of formula I means a branched or straight chain bivalent carbon radical containing 1-6 carbon atoms, such as methylene, ethylene (1,2-ethandiyl), propylene (1-methyl-1,2-ethanediyl), 2-methyl-1,2-ethanediyl, 2,2-dimethyl-1,2-ethanediyl, 1,3-propanediyl, 1,4-butanediyl, 1,5-pentanediyl and 1,6-hexanediyl.
The term phenylene means a bivalent moiety the free valencies of which can be positioned either ortho, meta or para to one another.
The term (C1-3)alkyl means a branched or straight chain alkyl group containing 1-3 carbon atoms, i.e. methyl, ethyl, propyl and isopropyl.
The term carboxyphenyl means a phenyl group which is substituted at either the ortho-, the meta- or the para-position with a carboxy-group. The ortho-carboxyphenyl group is preferred.
Compounds according to formula I wherein n+m is 7 are derivatives of xcex2-cyclodextrin, those wherein n+m is 8 are derived from xcex3-cyclodextrin.
Preferred are the 6-mercapto-cyclodextrin derivatives of formula I wherein X is COOH, or a pharmaceutically acceptable salt thereof.
More preferred are the 6-mercapto-xcex3-cyclodextrin derivatives of formula I wherein n is 8, R is (C1-6)alkylene and X is COOH.
Particularly preferred 6-mercapto-cyclodextrin derivatives of the invention are
6-per-deoxy-6-per-(2-carboxyethyl)thio-xcex3-cyclodextrin;
6-per-deoxy-6-per-(3-carboxypropyl)thio-xcex3-cyclodextrin;
6-per-deoxy-6-per-(4-carboxyphenyl)thio-xcex3-cyclodextrin;
6-per-deoxy-6-per-(4-carboxyphenylmethyl)thio-xcex3-cyclodextrin;
6-per-deoxy-6-per-(2-carboxypropyl)thio-xcex3-cyclodextrin; and
6-per-deoxy-6-per-(2-sulfoethyl)thio-xcex3-cyclodextrin.
The 6-mercapto-cyclodextrin derivatives of formula I can be prepared by reacting a C6-activated cyclodextrin derivative of formula II with an alkylthiol, arylalkylthiol or arylthiol derivative corresponding to Hxe2x80x94Sxe2x80x94Rxe2x80x94X, wherein R and X have the meaning as previously defined, in the presence of an inorganic or organic base. 
Formula II wherein m is 0-7, n is 1-8, m+n=7 or 8 and Y is a leaving group which can be a halide (Cl, Br or I), sulfuric ester or a sulfonic ester function, such as a tosylate, a napthtalenesulfonate or a triflate.
Conversely the 6-mercapto-cyclodextrin derivatives of formula I can also be prepared by reacting a 6-thiol xcex3- or xcex2-cyclodextrin derivative of formula III with an alkylating agent, e.g., alkyl halide, arylalkyl halide, alkyl sulfonate, arylalkyl sulfonate, corresponding to Yxe2x80x94Xxe2x80x94R, wherein Y, X and R have the meanings as previously defined, or with a double bond containing reagent, e.g., vinyl alkane, acrylate, etc., or an epoxide in the presence of an inorganic or organic base. 
Formula III wherein m is 0-7, n is 1-8, m+n=7 or 8.
Alternative synthesis routes for the preparation of the 6-mercapto-cyclodextrin derivatives of the invention are known to the skilled person. The chemistry of the derivatisation of cyclodextrins is well documented (see for example: Comprehensive Supramolecular Chemistry, Volumes 1-11, Atwood J. L., Davies J. E. D., MacNicol D. D., Vogtle F., eds; Elsevier Science Ltd., Oxford, UK, 1996).
Pharmaceutically acceptable salts of 6-mercapto-cyclodextrin derivatives of formula I wherein X represents the carboxylic acid group COOH, the sulphonic acid group SO2OH, the, phosphonic acid group PO(OH)2 or the tetrazol-5-yl group, may be obtained by treating the acid with an organic base or a mineral base, like sodium-, potassium- or lithium hydroxide.
The 6-mercapto-cyclodextrin derivatives, or pharmaceutically acceptable salts or solvates thereof, for use in the invention are administered parenterally. The injection route can be intravenous, subcutaneous, intradermal, intramuscular, or intra-arterial. The intravenous route is the preferred one. The exact dose to be used will necessarily be dependent upon the needs of the individual subject to whom the medicament is being administered, the degree of muscular activity to be restored and the judgement of the anaesthetist/critical-care specialist. Exfracorporal application of the chemical chelators of the invention, for instance by mixing of the chemical chelator with the blood during dialysis or during plasmapheresis, is also contemplated.
In a further aspect the invention relates to a kit for providing neuromuscular block and its reversal comprising (a) a neuromuscular blocking agent, and (b) a 6mercapto-cyclodextrin derivative according to general formula I capable of forming a guest-host complex with the neuromuscular blocking agent. With a kit according to the invention is meant a formulation, which contains separate pharmaceutical preparations, i.e. the neuromuscular blocking agent and a 6-mercapto-cyclodextrin derivative of formula I, i.e. the reversal agent. The components of such a kit of parts are to be used sequentially, i.e. the neuromuscular blocking agent and a 6-mercapto-cyclodextrin derivative of formula I, i.e. the reversal agent. The components of such a kit of parts are to be used sequentially, i.e. the neuromuscular blocking agent is administered to a subject in need thereof, which is followed, at a point in time when restoration of muscle function is required, by the administration of the reversal agent, i.e. a 6-mercapto-cyclodextrin derivative of the present invention.
A preferred kit, according to the invention, contains a 6-mercapto-cyclodextrin derivative of formula I and a neuromuscular blocking agent which is selected from the group consisting of rocuronium, vecuronium, pancuronium, rapacuronium, mivacurium, atracurium, (cis)atracurium, tubocurarine and suxamethonium. A particularly preferred kit of the invention comprises rocuronium as the neuromuscular blocking agent.
Mixed with pharmaceutically suitable auxiliaries and pharmaceutically suitable liquids, e.g. as described in the standard reference, Gennaro et al., Remington""s Pharmaceutical Sciences, (18th ed., Mack Publishing Company, 1990, Part 8: Pharmaceutical Preparations and Their Manufacture; see especially Chapter 84 on xe2x80x9cParenteral preparationsxe2x80x9d, pp. 1545-1569; and Chapter 85 on xe2x80x9cIntravenous admixturesxe2x80x9d, pp. 1570-1580) the 6-mercapto-cyclodextrin derivatives can be applied in the form of a solution, e.g. for use as an injection preparation.
Alternatively, the pharmaceutical composition may be presented in unit-dose or multi-dose containers, for example sealed vials and ampoules, and may be stored in a freeze dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example, water prior to use.
The invention further includes a pharmaceutical formulation, as hereinbefore described, in combination with packaging material suitable for said composition, said packaging material including instructions for the use of the composition for the use as hereinbefore described.